In making a diagnosis, pathologists often rely on preparation of the cytology or histopathology specimens being examined with one or a combination of several standard staining regimens. The most common of these are transmission stains (including Hematoxylin and Eosin (H&E) and PAP stains), immunohistochemical stains (DAB being the most common), and a variety of fluorescence probes targeted to proteins or structures outside of the nucleus, or to nucleic acid probes or genetic markers within the nucleus. As known in the art, H&E staining is one of the most common stains used in histology. This staining method involves application of the basic dye haematoxylin, which colors basophilic structures with blue-purple hue, and alcohol-based acidic eosin Y, which colors eosinophilic structures bright pink. There are numerous applications where it would be desirable to detect a plurality of (e.g. four or more) of these fluorescence probes within a single nucleus or cell in addition to being able to examine the morphology and architecture of the same specimen on the same histology slide using conventional transmission staining, such as H&E or pap.
Devices in which an acoustic beam and an optical beam interact are generally referred to as “acousto-optic devices” or AO devices. Certain AO devices are used for imaging. One example of an AO device for imaging is an AOTF. As well known in the art, the AOTF is a solid-state electronically tunable spectral bandpass filter, which operates on the principle of acousto-optic interaction in an anisotropic medium. The AOTF utilizes an anisotropic, birefringent AO medium for its operation. AOTFs are currently used in certain medical applications.
An AOTF is frequently used when it is desired to rapidly select one or more optical wavelengths from an incident optical beam containing a range of optical wavelengths. FIG. 1 shows the k-space diagram and accompanying real-space diagram for a typical non-collinear AOTF 100. The AOTF 100 shown includes a piezoelectric acoustic transducer t 105 bonded to a crystal of suitable birefringent AO interaction crystalline material 110 (PQWS in FIG. 1). The electrodes disposed on both sides of the transducer (t) 105 are not shown for simplicity. The phase velocity of the acoustic wave emerging from t is at an angle θa to the optical axis of the AO interaction crystal 110. Light enters the AOTF 100 through the input face RS of the AO interaction crystal 110 having incident polar angle θ, and wave vector k. Provided the RF frequency applied is adjusted to satisfy the resonant condition, the light is strongly diffracted to emerge through the output face PQ of the AO interaction crystal 110 with the diffracted wave vector kd, at a polar angle θd. The angular distance between the incident light and the first order diffracted light is therefore θd-θ, allowing separation of the respective light beams.
If white or other broadband light is incident on the AOTF, then once the RF frequency (and hence the acoustic wavelength and acoustic k-vector KA) is chosen, only a narrow band of optical wavelengths close to the resonant wavelength will be diffracted. Such a device may for example be used in a spectroscopic instrument or hyperspectral imaging system, where the input beam of unfiltered light corresponds to some part of the optical train, e.g. the “infinity space” of an optical microscope in which the angular divergence of the light collected from the object is purposely kept small. This infinity space is designed for the insertion of optical filters, polarizers and other components by the manufacturer. Although an optical microscope is used herein for the purposes of illustration, these comments apply equally well to any other optical imaging system. An AOTF has significant advantages for such an application because of its ability to tune very quickly between wavelengths. Typical non-collinear AOTFs made using TeO2 use acoustic wavelengths in the range of 5 to 20 μm in the interaction medium. By applying more than one RF frequency simultaneously, multiple optical pass bands may be created, this being an advantage besides speed provided by an AOTF over competing technologies.
Since AOTFs were developed many years before interest in precision applications including imaging reached its present high levels, conventional AOTFs do not provide the performance required for more demanding applications. In precision applications it is important for the AOTF to allow through only light wavelengths that are in a narrow band centered on the selected wavelength, and strongly reject all other wavelengths.
A standard AOTF uses continuous electrodes on both sides of the transducer. Such a device generates a uniform acoustic field throughout the interaction length in the AO interaction crystal. This arrangement that is not conducive to either high quality image formation or good filter response. A uniform acoustic field produces an optical pass-band that has poor rejection of adjacent wavelengths owing to the presence of significant sidelobes in the transmission function of the filter. It is the height of these sidelobes relative to the height of the main filter peak, which to a large extent determines the overall performance of the AOTF.
FIG. 2 shows an AOTF 200 including a rectangular transducer electrode 210 and associated AO interaction crystal 215, along with the resulting acoustic field shown below in the AO interaction crystal 215. The area of electrode 210 defines the functional area of the generally larger area associated transducer (transducer not shown in FIG. 2). It is generally known to improve AOTF operation the acoustic intensity in the interaction region of the AO interaction crystal should start off at a low level at the input end of the interaction region, then build up smoothly to a maximum in the center of the interaction region (x=L/2 in FIG. 2). After the center the acoustic intensity should fall off so that it is again low at the output end of the interaction region. The exact form of the mathematical function, which defines the rise and fall of the acoustic amplitude, S(x) in FIG. 2, is not unique. Many “windowing functions” have been used in the context of Fourier transforming of data, for example, the Parzen window, the cosine window, the truncated Gaussian, and most are generally suitable.
The local strength of the acoustic wave generated by a piezoelectric transducer depends on the product of (1) the local electric field strength and (2) the local piezoelectric activity, the latter being related to the crystal structure of the transducer. Usually the transducer used in acousto-optic devices is a single crystal of lithium niobate (LN). A piezoelectric material such as LN is a so-called “hard ferroelectric” and it is difficult to manipulate the local piezoelectric strength in the way one may for example manipulate the local piezoelectricity of a piezoelectric ceramic. This latter material being typically used in acoustic transducers for generation of lower frequency (tens to hundreds of kHz) acoustic waves for example in sonar applications. Thus, it is relatively easy to arrange for a sonar transducer launching an acoustic beam into water to be apodized by controlling the degree of local poling of the piezoelectric material, and so generate an acoustic beam of arbitrary spatial intensity distribution. However, it is comparatively difficult to apodize the beam from a LN transducer launching an acoustic beam into an AO crystal. A designer generally has only two options in practice; to attempt alter the piezoelectric activity, or to locally alter the electric field strength.
Local alteration of the electric field inside the LN crystal comprising the acoustic transducer can be achieved by patterning the top electrode. Instead of a continuous top electrode of substantially rectangular form which is conventionally used, a pattern can be chosen which achieves a gradual reduction of the average electric field in the piezoelectric transducer with movement out towards the input and output faces of the acousto-optic interaction crystal, and thus generates some apodization of the acoustic wave. This has been achieved by dividing up the rectangular top electrode into a small number (e.g. up to 11) of electrically independent sub-electrodes and driving at least some of the electrodes using a multiplicity of independent RF drivers. In this arrangement, each electrode segment requires its own matching circuit and flexible cable connection to the multichannel “driver”, the latter containing all the RF drive electronics including the RF amplifiers. All the cables must be closely matched in length to within a few mm and the adjustment procedure needed to get all the transducer sub-elements operating substantially in phase over the whole tuning range is difficult, requiring a high level of skill. If the transducer elements do not operate in phase due to incorrect adjustment or manufacture, the device will not work properly. The RF power fed to the transducer sub-elements is chosen to approximate to the desired apodization function, for example a Gaussian, the “tails” being at the input and output ends of the interaction region, and the maximum being in the center. This method generally works well when it is adjusted properly, and side-lobe reductions of 20 dB can be achieved, however it is complicated and expensive, requiring a complicated and expensive bank of RF drivers rather than a single RF driver.
It is a known peculiarity of the AOTF that the spatial resolution, as well as the spectral resolution, is determined by the spectral sidelobe rejection within the AOTF. The quality the imaging provided by the AOTF thus generally relates to the magnitude of the sidelobes transmitted along with the beam of interest.
Regarding pathology, a pathologist now generally requires a separate tissue section or cytology specimen for each target to be examined with a fluorescence probe, and an additional specimen for the transmission stain examination. Examination of these numerous slides serve as multiple adjuncts for the pathologist in making the final diagnosis. What is needed is a new AOTF design that strongly shapes or “apodizes” the acoustic beam to optimize filter quality and image quality by substantially reducing the level of the undesired sidelobes transmitted with the desired optical beam. An AOTF-based system providing sharply reduced side lobes would permit simultaneously imaging a plurality fluorescent or transmission tags, such as samples prepared with both a transmission stain and numerous fluorescence markers, for improved hyperspectral and multispectral imaging of medical tissues. Such a system would allow all these tests to be performed on a single slide with a single sample, thereby minimizing the amount of tissue required, avoiding artifacts resulting from the use of different parts of the tissue being used for each slide, and simplifying and speeding-up interpretation by the pathologist.